The following description provides a summary of information relevant to the present invention and is not a concession that any of the information provided or publications referenced herein is prior art to the presently claimed invention.
An assay can be performed in a number of ways such as the well-known sandwich technique and competitive technique. A variety of specific biological binding molecules can be labeled with a radioactive element, a fluorophore or a constituent which enters into an enzyme reaction. Thus a sample containing suspected target ligands can be analyzed, and the target ligand or target ligands can be detected quantitatively by forming a complex with a labeled anti-ligand, labeled as indicated above, and measuring the labeled constituent in the complex to determine the quantity of the target ligand. The anti-ligand binds to at least one site on the ligand to form a complex. “Ligand or target ligand” and “anti-ligand,” as these terms are used herein, refer to antigens, antibodies, binding proteins, haptens, hormone receptors, and other biological molecules that can form a complex. The labeled anti-ligand used to detect and measure the target ligand in the complex is referred to herein as the “detector ligand.”
In the sandwich technique mentioned above, a target ligand, a detector ligand, and a capture ligand are used in the assay. The detector ligand is detected quantitatively in a detector ligand/target ligand complex to determine a quantity of the target ligand present. The capture ligand in this technique is an anti-ligand that binds to the target ligand. The capture ligand and detector ligand typically bind to different sites on the target ligand so that there is no interference between the binding of the detector ligand to the target ligand and the capture ligand to the target ligand.
In the sandwich technique, the target ligand binds to the capture ligand to form a first complex. The detector ligand also binds to the target ligand in the complex to form a second complex in the sandwich, and the labeled constituent in the sandwiched ligands is detected quantitatively to deduce the quantity of target ligand present. Detection can be performed by: measuring radioactivity where the detector ligand is radioactive; measuring fluorescent light where there is a fluorescent label on the detector ligand; or spectrophotometrically where an optical density or wavelength change occurs through an enzyme reaction, or through fluorescent quenching. Detection may require separation of the sandwiched ligands from unbound ligands and this is generally done by separating the capture ligand attached or immobilized onto a surface from a solution containing unbound detector ligands.
The quantity of target ligand is deduced from the quantity of detector ligand detected, because the two quantities are generally directly proportional to each other in the sandwich technique. Parallel tests against known standards are employed for calibration.
The quantity of target ligand can be determined as an inverse proportion using the competitive technique previously mentioned above. With this technique, the capture ligand is contacted either simultaneously or sequentially with a target ligand and a known, limiting quantity of detector ligand. When the target ligand and detector ligand bind to the capture ligand, the quantity of detector-ligand detected in a binary complex with the capture ligand is inversely proportional to the amount of target ligand present. In this technique, the target ligand and detector ligand bind to the same site or sites in close proximity to each other to create competition.
The two techniques discussed above may be represented as follows where C designates the capture ligand, T represents the target ligand, and D represents the detector ligand.
SandwichCompetitive1. C + T → CT1. C + T → CT + Csequential2. CT + D → CTD2. (CT + C) + D → CT + CD1. C + T + D → CD + CTsimultaneous
In addition to the sandwich and competitive assays described above, a variety of other techniques for assays are known, and include the following assays.
A method of using two different ligands tagged with two different tagging constituents in an immunoassay to independently detect and measure bound target ligand and bound receptor or capture ligand is described in U.S. Pat. No. 4,385,126 to James H. Chen et al.
A method of using oligonucleotides as capture agents for capture ligands in assays is described in U.S. Pat. No. 5,648,213 to Reddy et al.
Assay reagents and kits using oligonucleotides as capture agents in an assay array is described in U.S. Pat. No. 5,789,165 to Oku et al.
Although these systems are useful, there is always a need for systems with improved accuracy and lower costs. Accordingly, a need exists for a system that: offers greater precision in detection and quantification of capture ligands; offers greater precision in detection and quantification of target ligands; decreases variation arising from imprecision in the addition of reagents; offers correction for the variation arising from various assay manipulations; decreases the amount of binding interference caused by random attachment or positioning of a label on capture ligands; decreases the variability between different preparations of the same labeled capture ligands; provides reusable components to decrease expense in doing multiple assays; provides easy quality control and standardization methods; and can be provided as an assay kit.